3.1. Collagens and Disorders of Collagen Metabolism
Osteoporosis is the most common bone disease in humans. Primary osteoporosis, accompanied by increased susceptibility to fractures, results from a progressive reduction in skeletal bone mass. It is estimated to affect 15–20 million individuals in the USA alone. Its basis is an age-dependant imbalance in bone remodelling, i.e. in the rates of formation and resorption of bone tissue.
In the USA about 1.2 million osteoporosis-related fractures occur in the elderly each year including about 538,000 compression fractures of the spine, about 227,000 hip fractures and a substantial number of early fractured peripheral bones. Between 12 and 20% of the hip fractures are fatal because they cause severe trauma and bleeding, and half of the surviving patients require nursing home care. Total costs from osteoporosis-related injuries now amount to at least $10 billion annually in the USA (Riggs, New England Journal of Medicine, 327:620–627 (1992)).
Osteoporosis is most common in postmenopausal women who, on average, lose 15% of their bone mass in the 10 years after menopause. This disease also occurs in men as they get older and in young amenorrheic women athletes. Despite the major, and growing, social and economic consequences of osteoporosis, the availability of reliable assays for measuring bone resorption rates in patients or in healthy subjects is very limited. Other disorders entailing (and correlated with) abnormalities in collagen metabolism include Paget's disease, Marfan's syndrome, osteogenesis imperfecta, neoplastic growth in collagenous tissue, dwarfism, rheumatoid arthritis, osteo-arthritis and vasculitis syndrome.
Three known classes of human collagen have been described to date. The Class I collagens, subdivided into types I, II, III, V, and XI, are known to form fibrils.
The amino-acid sequence of type I–III (to the extent it has been elucidated) is given in Appendix A of WO 95/08115.
Collagen type I accounts for more than 90% of the organic matrix of bone. Therefore, in principle, it is possible to estimate the rate of bone resorption by monitoring the degradation of collagen type I. Likewise, a number of other disease states involving connective tissue can be monitored by determining the degradation of collagen. Examples are collagen type II degradation associated with rheumatoid arthritis and osteo-arthritis and collagen type III degradation in vasculitis syndrome.
Amino acid sequences of human type III collagen, human pro al(II) collagen, and the entire prepro al(III) chain of human type III collagen and corresponding cDNA clones have been investigated and determined by several groups of researchers; see Loil et al., Nucleic Acid Research 12:9383–9394 (1984): Sangiorgi et al., Nucleic Acids Research, 13:2207–2225 (1985); Baldwin et al., Biochem J., 262:521–528 (1989); and Ala-Kokko et al., Biochem. J., 260:509–516 (1989).
Type I, II, and III collagens are all formed in the organism as procollagen molecules, comprising N-terminal and C-terminal propeptide sequences, which are attached to the core collagen molecules. After removal of the pro-peptides, which occurs naturally in vivo during collagen synthesis, the remaining core of the collagen molecules consists largely of a triple-helical domain having terminal telopeptide sequences which are non-triple-helical. These telopeptide sequences have an important function as sites of intermolecular cross-linking of collagen fibrils extra-celluarly. The alpha-helical region also includes cross-linkable sites.
Intermolecular cross-links provide collagen fibrils with biomechanical stability. The formation of these cross-links is initiated by modification of lysine and hydroxylysine residues to the corresponding aldehydes. Several of these residues located on adjacent chains of collagen will spontaneously form different intermolecular cross-links. The exact position of the sites for cross-linking on collagen telopeptides and from the helical region has been previously described. See, for example, Kühn, K., in Immunochemistry of the extracellular matrix, 1:1–29, CRC Press, Inc., Boca Raton, Fla. (1982), Eyre, D. R., Ann. Rev. Biochem., 53:717–48 (1984) or U.S. Pat. Nos. 5,140,103 and 5,455,179. Furthermore, the amino acid sequences of some potential sites for cross-linking in type I, II, and III collagen are given in Table 1 below.
The fibrous proteins, collagen and elastin, are cross-linked by a unique mechanism based on aldehyde formation from lysine or hydroxylysine side chains. Four homologous loci of cross-linking are evident in molecules of type I, II and III collagens (for review see Kühn, K., in Immunochemistry of the extracellular matrix, 1:1–29 (1982)). Two are aldehyde sites, one in each telopeptide region. The other two sites are hydroxylysine symmetrically placed at about 90 residues from each end of the molecule. When collagen molecules pack into fibrils, these latter sites in the helical region align and react with telopeptide aldehydes in adjacent molecules. There is now strong evidence that 3-hydroxypyridinium residues are the mature cross-link coming from hydroxylysine-derived aldehydes. The mature cross-linking residues of the other pathway, i.e. from aldehyde formation of lysine residues, are however, still unknown.
As illustrated by formula in EP-0394296 discussed below, the two 3-hydroxypyridinium cross-links have been found to be hydroxylysyl pyridinoline (also known simply as “pyridinoline”) and lysyl pyridinoline (also known as “deoxypyridinoline”). These cross-linking compounds are naturally fluorescent. Some hydroxylysyl pyridinoline cross-link are found to be glycosylated as discussed for instance in EP-A-0424428.
However, as described in Last et al, Int. J. Biochem. Vol. 22, No. 6, pp 559–564, 1990 other crosslinks occur naturally in collagen.
3.2. Prior Art Assays for Collagen Degradation
In the past, assays have been developed for monitoring degradation of collagen in vivo by measuring various biochemical markers, some of which have been degradation products of collagen.
For example, hydroxyproline, an amino acid largely restricted to collagen, and the principal structural protein in bone and all other connective tissues, is excreted in urine. Its excretion rate is known to be increased in certain conditions, notably Paget's disease, a metabolic bone disorder in which bone turnover is greatly increased, as discussed further below.
For this reason, urinary hydroxyproline has been used extensively as an amino acid marker for collagen degradation; Singer, F. R. et al., Metabolic Bone Disease, Vol. II (eds. Avioli, L. V., and Kane, S. M.), 489–575 (1978), Academic Press, New York.
U.S. Pat. No. 3,600,132 discloses a process for the determination of hydroxyproline in body fluids such as serum, urine, lumbar fluid and other intercellular fluids in order to monitor deviations in collagen metabolism. The Patent states that hydroxyproline correlates with increased collagen anabolism or catabolism associated with pathological conditions such as Paget's disease, Marfan's syndrome, osteogenesis imperfecta, neoplastic growth in collagen tissues and in various forms of dwarfism.
Bone resorption associated with Paget's disease has also been monitored by measuring small peptides containing hydroxyproline, which are excreted in the urine following degradation of bone collagen; Russell et al., Metab. Bone Dis. and Rel. Res. 4 and 5, 2250262 (1981), and Singer, F. R., et al., supra.
In the case of Paget's disease, the increased urinary hydroxyproline probably comes largely from bone degradation; hydroxyproline, however, generally cannot be used as a specific index for bone degradation. Much of the hydroxyproline in urine may come from new collagen synthesis (considerable amounts, of the newly made protein are degraded and excreted without ever becoming incorporated into tissue fabric), and from turnover of certain blood proteins as well as other proteins that contain hydroxyproline.
Furthermore, about 80% of the free hydroxyproline derived from protein degradation is metabolised in the liver and never appears in the urine. Kiviriko, K. I., Int. Rev. Connect. Tissue Res. 5:93 (1970), and Weiss, P. H. and Klein, L., J. Clin. Invest. 48:1 (1969). Hydroxyproline is a good marker for osteoporosis as it is specific for collagen in bones even if it is not specific for bone resorption, but it is trouble-some to handle.
Hydroxylysine and its glycoside derivatives, both peculiar to collagenous proteins, have been considered to be more accurate than hydroxyproline as markers of collagen degradation. However, for the same reasons described above for hydroxyproline, hydroxylysine and its glycosides are probably equally non-specific markers of bone resorption; Krane, S. M. and Simon, L. S., Develop. Biochem. 22:185 (1981).
Other researchers have measured the cross-linking compound 3-hydroxypyridinium in urine as an index of collagen degradation in joint diseases. See, for background and as examples, Wu and Eyre, Biochemistry, 23:1850 (1984): Black et al., Annals of the Rheumatic Diseases, 45:969–973 (1986); and Seibel et al., The Journal of Dermatology, 16:964 (1989). In contrast to the present invention, these prior researchers have hydrolysed peptides from body fluids and then looked for the presence of free 3-hydroxypyridinium residues.
Assays for determination of the degradation of type I, II, and III collagen are disclosed in EP-0394296 and U.S. Pat. No. 4,973,666 and U.S. Pat. No. 5,140,103. However, these Patents are restricted to collagen fragments containing the cross-linker 3-hydroxypyridinium. Furthermore, the above mentioned assays require tedious and complicated purifications from urine of collagen fragments containing 3-hydroxypyridinium to be used for the production of antibodies and for antigens in the assays.
Until recently very few clinical data using the approach described in U.S. Pat. No. 4,973,666 and U.S. Pat. No. 5,140,103 are available. Particularly, no data concerning the correlation between the urinary concentration (as determined by methods described in the above mentioned patents) of 3-hydroxypyridinium containing telopeptides of type I collagen and the actual bone loss (as determined by repeated measurements by bone densitometry) had been published. Very recently however McClung et al (JBMR (1996) 11:129) have concluded that results from the commercial NTx assay based on these Patents do not correlate to bone loss. More particularly, NTx did not correlate to bone loss in the normal population and also failed to predict bone changes in response to therapy. Gertz et al (JBMR (1994) 9(2): 135–142) have reported no significant correlation between baseline NTx measurements and bone loss and no significant correlation between change in NTx and change in bone loss during anti-resorptive therapy.
Garnero et al (JBMR (1996) 11(10): 1531–1537) have reported that NTx was found not be predictive of hip fracture whilst other biochemical markers were associated with an approximately 100 percent increased risk of hip fracture.
The presence of 3-hydroxypyridinium containing telopeptides in urine requires the proper formation in bone tissue of this specific cross-linking structure at various times before the bone resorbing process. Very little information on these processes is available and it would be desirable to avoid this dependence of the correct formation of the cross-linking structure.
GB Patent Application No. 2205643 reports that the degradation of type III collagen in the body can be quantitatively determined by measuring the concentration of an N-terminal telopeptide from type III collagen in a body fluid. This method uses antibodies generated to N-terminal telopeptides released by bacterial collagenase degradation of type III collagen, said telopeptides being labelled and used in the assay.
Schrater-Kermani et al., Immunol. Invest. 19:475–491 (1990) describe immunological measurement systems based on CNBr fragments of collagen type I and II. Use is made of pepsin-solubilised collagen, leaving the telopeptides in the tissue (cf. the above mentioned GB Patent Application No. 2205643). There is therefore no conformity between the fragments and the antibodies raised therefrom. Further, the reference only describes measurements on extracted tissue samples.
The development of a monoclonal antibody raised against pepsin-solubilised type I collagen is described in Werkmeister et al., Eur. J. Biochem. 1987:439–443 (1990). The antibody is used for immunohistochemical staining of tissue segments and for measuring the collagen content in cell cultures. The measurements are not carried out on body fluids.
EP Patent Application No. 0505210 describes the development of antibody reagents by immunisation with purified cross-linked C-terminal telopeptides from type I collagen. The immunogen is prepared by solubilising human bone collagen with bacterial collagenase. The antibodies thus prepared are able to react with both cross-linked and non-cross-linked telopeptides, and cross-linkers other than pyridinoline.
There are a number of reports indicating that collagen degradation can be measured by quantitating certain pro-collagen peptides. Propeptides are distinguished from telopeptides and alpha-helical region of the collagen core by their location in the procollagen molecule and the timing of their cleavage in vivo; see U.S. Pat. No. 4,504,587; U.S. Pat. No. 4,312,853; Pierard et al., Analytical Biochemistry 141:127–136 (1984); Niemela, Clin. Chem. 31/8:1301–1304 (1985); and Rohde et al., European Journal of Clinical Investigation, 9:451–459 (1979).
EP Patent Application No. 0298210 and No. 0339443 both describe immunological determination of procollagen peptide type III and fragments thereof. Further, a method based on the measurement of procollagen is disclosed in EP Patent Application No. 0465104.
The use of synthetic peptides with sequences derived from type IX collagen for the development of immunological reagents is disclosed in PCT Patent Application No. WO 90/08195. Likewise the application describes the use of the antibodies thus produced for the determination of type IX collagen fragments in body fluids. U.S. Pat. No. 4,778,768 relates to a method of determining changes occurring in articular cartilage involving quantifying proteoglycan monomers or antigenic fragments thereof in a synovial fluid sample.
Dodge, J. Clin Invest 83:647–661 (1981) discloses methods for analysing type II collagen degradation utilising a polyclonal antiserum that specifically reacts with unwound alpha-chains and cyanogen bromide-derived peptides of human and bovine type II collagens. The degradation products of collagen were not detected in a body fluid, but histochemically by staining of cell cultures, i.e. by “in situ” detection.
WO 94/03813 describes a competitive immunoassay for detecting collagen or collagen fragments in a sample wherein a binding partner containing a synthetic linear peptide corresponding to the non-helical C-terminal or N-terminal domain of collagen is incubated with an antibody to the linear synthetic peptide and the sample, and wherein the binding of the antibody to the binding partner is determined.
WO 95/08115 relates to assay methods in which collagen fragments in a body fluid are determined by reaction with an antibody which is reactive with a synthetic peptide. The assay may be a competition assay in which the sample and such a peptide compete for an antibody, possibly a polyclonal antibody raised against fragments of collagen obtained by collagenase degradation of collagen. Alternatively, it may be an assay in which an antibody, possibly a monoclonal anti-body, is used which has been raised against such a synthetic peptide.
As disclosed in WO 91/08478, one particular type of peptide fragment found in body fluid, particularly urine, is of the formula:

In the above formula, K-K-K is disclosed as representing a hydroxypyridinium cross-link but in fact it may be any naturally occurring cross-link and specifically any of those discussed in the above referenced paper of Last et al. As further discussed below, larger peptide fragments including the above smaller fragment are also disclosed in this document.
A proportion of the “peptide” fragments in body fluid are related to peptides of equivalent amino acid sequence, e.g. peptides of formula 1, by the isomerisation of aspartic acid in the formula to isoaspartic acid. We put “peptides” in quotes here as of course the isomerisation means that these species are no longer properly regarded as being peptides.
The isomerisation of proteins containing aspartic acid has been reported previously to be a spontaneous reaction occurring under physiological conditions.
See for instance Brennan et al Protein Science 1993, 2, 331–338, Galletti et al, Biochem. J. 1995, 306, 313–325, Lowenson et al, Blood Cells 1988, 14, 103–117 and Oliya et al, Pharmaceutical Research, Vol. 11, No. 5, 1994, p. 751.
The isomerisation has the effect of transferring that part of the peptide chain which runs downstream of the aspartic acid residue in the carboxy terminus direction from the alpha carboxylic acid of the aspartic acid to which it is bonded via a peptide bond in the normal protein to the side chain carboxylic acid in a non-peptide amide bond, as shown below:

The non-peptide bonded aspartic acid residue is termed “isoaspartic acid” or β-aspartic acid (βD)
Similar isomerisation can occur in proteins containing asparagine residues (i.e. with —NH2 instead of —OH in the starting protein in the above reaction scheme).
The above discovery indicates that this isomerisation also occurs in bone tissue and the extent of isomerisation is expected therefore to be marker for the age of the bone tissue concerned.
Furthermore, the presence amongst such bone peptide fragments of the isomerised peptides provides confirmation that the fragments indeed derive from bone degradation and not some other source such as the degradation of newly formed collagen never incorporated into bone.
J. Macek and M. Adam “Determination of collagen degradation products in human urine”, Z. Rheumatol. 46:237–240 (1987) reports the presence of pyridinoline containing collagen cross-linked peptides in urine having a molecular weight above 10,000 but provides no sequence information relating to the peptide chains present or the collagen type to which the fragments belong.
As mentioned above WO91/08478 discloses that a number of fragments of type 1 collagen can be found in urine. These include a pyridinium crosslink, which may be hydroxylysyl pyridinoline or lysyl pyridinoline. Attached to the crosslink are peptide chains of defined sequence derived from the collagen molecule. The crosslink has three points at which it may bear peptide chains. The fragment of Formula 1 above (Formula VI in WO91/08478) bears two chains, each having the sequence EKAHDGGR. Two other fragments are described in WO91/08748 which each have a third chain, that shown in Formula IV of that specification being 7 amino acids longer than that in Formula V, but otherwise of the same sequence. The amino-acid sequence of the chains of type 1 collagen has been published elsewhere as described above, as has the location of the trivalent crosslinks between the collagen molecules. The third chain depicted in the said Formulae IC and V of WO91/08748 does not correspond to that of any collagen chain at the location of the crosslink and is believed to be an error, possibly caused by an artefact of the isolation procedure used.
The only fragment for which a credible formula has been given is there for that of Formula VI (equivalent to Formula 1 herein) having two identical peptide chains.
DE-A-4225038 discloses sandwich assays for collagen breakdown products in body fluids. Antibodies are to be produced by immunisation with haptens containing a linear sequence of amino acids. One proposed sequence is FDFSFLP (SEQ ID No.2) and another is PPQEKAHDGGR (SEQ ID No.3), although these were not suggested for use in combination to make two antibodies for use in the same sandwich assay. Indeed although the sequence PPQEKAHDGGR is given, no antibodies made against it are specifically described and therefore no disclosure is provided of their actual utility and properties. The only specific sandwich assay described combines an antibody against the C-terminal sequence FDFSFLP with one against the sequence GMKGHRGF (SEQ ID No.4) (from the helical region crosslink site).
However, DE-A-4225038 asserts that there is a close correlation between results obtained using an assay based on the sequence FDFSFLP and a commercial assay known as the ICTP assay. It has been shown however that the ICTP assay in serum does not appear to reflect bone resorption in that the results it produces do not successfully track the effect of therapeutic treatment (Hassager et al. Calcif. Tissue. Int. (1994) 54:40–33). This of course would imply that the population of reflect bone resorption in a useful way.
We have now established that body fluids do in fact contain larger collagen fragments containing not only the sequence EKAHDGGR but also further amino acid residues. These may be present in a third chain attached to the crosslink with two chains incorporating the sequence EKAHDGGR and/or as extensions of N-amino terminal direction of the sequence EKAHDGGR of one or both of the two chains containing that sequence.
We have further established that surprisingly it is possible to obtain binding of two distinct antibodies to a single collagen degradation fragment where both antibodies are specific for an epitope in the sequence EKAHDGGR or a variant of it.